Mel'nikova, S. A. Konnova, and O. M. Abros'kina. Institute of Biochemistry and Physiology of Plants and Microorganisms,. Russian Academy of Sciences, pr.
Microbiology, Vol. 70, No. 4, 2001, pp. 451–455. Translated from Mikrobiologiya, Vol. 70, No. 4, 2001, pp. 519–524. Original Russian Text Copyright © 2001 by Karpunina, Mel’nikova, Konnova, Abros’kina.
EXPERIMENTAL ARTICLES
Role of the Agglutinating Proteins of Bacilli and Rhizobia in Bacterial Interactions L. V. Karpunina, U. Yu. Mel’nikova, S. A. Konnova, and O. M. Abros’kina Institute of Biochemistry and Physiology of Plants and Microorganisms, Russian Academy of Sciences, pr. Entuziastov 13, Saratov, 410015 Russia Received July 12, 2000
Abstract—The surface agglutinating proteins of the soil nitrogen-fixing bacteria Bacillus polymyxa 1460 and Rhizobium leguminosarum 252 were found to be able to interact with the polysaccharide complexes of certain azospirilla and rhizobia. Such interactions are most likely involved in the formation of nitrogen-fixing plantbacterial associations in the rhizosphere. Key words: agglutinating proteins, polysaccharides, nitrogen-fixing bacteria, interaction.
Data available in the literature on bacterial interactions are scarce and primarily deal with intrageneric interactions, such as the flocculation of rhizobia [1] and the aggregation of agrobacteria and bradyrhizobia on the root surface [2, 3]. At the same time, such data are of interest, especially as concerned with the nitrogenfixing rhizosphere bacteria and with the molecular mechanisms underlying bacterial cell recognition. Little is known about the interaction of microorganisms in mixed cultures, including nitrogen-fixing associations, although it would be reasonable to suggest that the surface components of bacterial cells, particularly lectins and polysaccharides, are involved in such interaction. The aim of the present work was to study the role of the surface agglutinating proteins of soil nitrogen-fixing bacilli and rhizobia in bacterial interactions. MATERIALS AND METHODS Experiments were performed with the lectins LI and LII of Bacillus polymyxa 1460 and the agglutinins R1 and R2 Rhizobium leguminosarum 252 of Rhizobium leguminosarum 252, which were stripped from the surface of bacterial cells as described earlier [4, 5]. The polysaccharide–lipid complex (PSLC) and lipopolysaccharide–protein complex (LPPC) of Azospirillum brasilense strains Sp7, Sp245, SR75, and S17 and Azospirillum lipoferum strains 59b and RG20a were isolated from the capsular material of these strains using chromatographic methods [6]. The exopolysaccharide (EPS) and lipopolysaccharide (LPS) of R. leguminosarum 252 were obtained from L.V. Kosenko, Zabolotnyi Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine in Kiev. The monosugar composition of the polysaccharidecontaining complexes of azospirilla was analyzed by thin-layer chromatography (TLC) on DC-Alufolien
cellulose plates in a pyridine–ethylacetate–acetic acid– water (5 : 5 : 1 : 3) solvent system. Separated monosugars were visualized by spraying the developed plates with a solution of anisidine phthalate in butanol. Samples for TLC were prepared by hydrolyzing polysaccharide complexes in 4 N trifluoroacetic acid (TFA) at 100°C for 4 h in sealed ampules; the TFA was then removed with a rotary vacuum pump [7]. The interaction of agglutinating proteins with the polysaccharide complexes was studied by the blotting technique [8], detecting immune complexes on nitrocellulose membranes with the protein A–gold colloid conjugate manufactured at the Institute of Biochemistry and Physiology of Plants and Microorganisms. The concentration of bacillary lectins and rhizobial agglutinins was 40 µg/ml and that of the polysaccharide complexes was 1 mg/ml. Positive reaction manifested itself as a pink spot on a nitrocellulose membrane. The specificity of interaction was studied by incubating bacillary lectins (40 µg/ml; 28°C; 30 min) with one of the specific haptens (such as 0.3 M glucuronic acid) and by incubating rhizobial agglutinins (40 µg/ml) with their antibodies. The blocking of hemagglutinating activity was evaluated by assaying this activity with trypsinized rabbit erythrocytes. Antibodies to the lectins LI and LII of B. polymyxa 1460 and the agglutinins R1 and R2 of R. leguminosarum 252 were prepared as described earlier [4, 5]. RESULTS AND DISCUSSION As was shown in our previous works [4, 5, 9–11], the lectins of B. polymyxa 1460 and R. leguminosarum 252 are involved in the attachment of bacterial cells to host plants, binding to plant receptors of a protein or carbohydrate nature. The role of bacterial lectins in
0026-2617/01/7004-0451$25.00 © 2001 åÄIä “Nauka /Interperiodica”
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KARPUNINA et al. (‡) LI LII R1 R2 (b) LI LII R1 R2 1
2
3
4
5
6
7
8
9
10
11
12
13 14
Fig. 1. Dot immunoblotting analysis of the interaction of the B. polymyxa 1460 lectins and R. leguminosarum 252 agglutinins with the polysaccharide-containing complexes of soil bacteria: (a) native lectins and agglutinins; (b) bacillary lectins blocked with glucuronic acid and rhizobial agglutinins blocked with antibodies; (1, 2, 3, and 4) PSLC; (5, 6, 7, and 8) the LPPC of A. brasilense Sp7, SR75, Sp245, and S17; (9 and 10) the EPS and LPS of R. leguminosarum 252; (11 and 12) the PSLC and LPPC of A. lipoferum 59b; and (13 and 14) the PSLC and LPPC of A. lipoferum RG20a.
intercellular interactions was not studied in depth, although there is evidence that they may be involved in microbial flocculation [12]. In the present work, we found that the bacillary lectins and rhizobial agglutinins are able to recognize receptors in some bacterial polysaccharide complexes: lectin LI, which is specific for glucuronic acid and fructose-1,6-diphosphate, and lectin LII, which is also specific for galacto- and glucosamine [4], interacted with all of the studied azospirillary PSLC and LPPC and rhizobial EPS and LPS.
EPS
LPS
Glucose Glucosamine Glucuronic acid
Glucose Fucose Rhamnose Mannose Galactose Glucuronic acid Galacturonic acid Glucosamine Quinovosamine
Fig. 2. Monosugar composition of the surface polysaccharides of R. leguminosarum 252.
Similarly, the rhizobial agglutinins interacted with both azospirillary and rhizobial polysaccharide complexes; in this case, the agglutinin R2 was much more active than the agglutinin R1 (Fig. 1). The blocking of the agglutinating activity of lectin LI with glucuronic acid partially inhibited its interaction with the PSLC and LPPC of A. brasilense Sp7, SR75, and Sp245 and A. lipoferum 59b and RG20a and with the EPS and LPS of R. leguminosarum 252, as follows from the decrease in the intensity of the pink color of the spots on the nitrocellulose filters (Fig. 1). The incomplete inhibition of the interaction is indicative of the involvement of specific and nonspecific mechanisms. The specific interaction of lectin LI with the rhizobial EPS and LPS can be due to the presence of glucuronic acid in these complexes [13] (Fig. 2). The specific interaction of lectin LI with the azospirillary polysaccharide complexes, which lack glucuronic acid and fructose-1,6-diphosphate (see table), can be explained by its interaction with 2-keto-3-deoxyoctonate (KDO) present in these complexes [6]. A similar effect was observed by Yagoda-Shagam et al. [14], who found that the sialic acid–specific lectin is able to interact with the capsular material of azospirilla, although this material lacks sialic acid. On the other hand, lectin LI was unable to interact with the polysaccharides of A. brasilense S17, presumably due to the fact that these polysaccharides lack carbohydrates specific for this lectin (see table). It should be noted that the polysaccharides of this strain differ from the polysaccharides MICROBIOLOGY
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Monosugar composition of the polysaccharide complexes of azospirilla Monosugar Strain A. brasi Sp7 lense Sp245 SR75 S17 A. lipo- RG20a ferum 59b
Complex Rhamnose Galactose Fucose LPPC PSLC LPPC PSLC LPPC PSLC LPPC PSLC LPPC PSLC LPPC PSLC
+ + + + + + + + +
+ + + + + + + + +
Xylose Mannose
+ + + +
+ + +
+ +
Glucosamine + + + + + + + + + + +
Galacto- GalactuGlucose samine ronic acid + + + + + +
+ + + + + + + +
+ + +
+ +
Note: The “+” sign indicates the presence of the particular monosugar.
of the other A. brasilense strains studied in the high mannose content (see table). The absence of the formation of pink spots on nitrocellulose membranes as a result of the incubation of the glucuronic acid–treated lectin LII with the PSLC of A. brasilense Sp245 and the LPPC of A. brasilense Sp7 is indicative of the specificity of the interaction of lectin LII with these polysaccharide complexes. This specific interaction may be due to glucosamine and galactosamine present in the PSLC (see table). On the other hand, lectin LII was unable to interact with the PSLC and LPPC of A. brasilense S17, although these complexes contain galacto- and glucosamine; this could be related to the steric inaccessibility of carbohydratebinding sites in these complexes. The incubation of lectin LII with the PSLC of A. brasilense Sp7, the LPPC and PSLC of A. brasilense SR75, the LPPC of A. brasilense Sp245, the PSLC and LPPC of A. lipoferum 59b and RG20a, and the EPS and LPS of R. leguminosarum 252 produced only faint pink spots on nitrocellulose filters; this suggests that the interaction of lectin LII with these polysaccharide complexes is due to both the specific haptens of these complexes and nonspecific binding (Figs. 1 and 2). Experiments on the blocking of the agglutinating activity of the rhizobial agglutinin R1 with the respective antibodies showed that this lectin interacts, both specifically and nonspecifically, with the LPPC of A. brasilense Sp7, SR75, and Sp245; the PSLC of A. brasilense S17; and rhizobial LPS. The interaction of agglutinin R1 with the PSLC of A. brasilense Sp7, SR75, and Sp245; the EPS of R. leguminosarum 252; the LPPC of A. brasilense S17; and the PSLC and LPPC of A. lipoferum 59b and RG20a was only nonspecific. Data on the interaction of agglutinin R1 with MICROBIOLOGY
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LPS are in good agreement with our earlier findings that haptens for this agglutinin may represent complex carbohydrates [13]. As for agglutinin R2, it was able to interact, both specifically and nonspecifically, with the PSLC and LPPC of A. brasilense S17 and the LPS of R. leguminosarum 252, whereas its interaction with the other polysaccharide complexes was only nonspecific (Fig. 1).
Fig. 3. Interaction of the agglutinin R1 of R. leguminosarum 252 with the LPPC of A. brasilense Sp7. The twofold agglutinin dilutions are in the downward direction and the twofold LPPC dilutions are in the right direction.
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LI
PSLK LPPC
A. brasilense Sp7
PSLC LPPC
A. brasilense SR75
PSLC LPPC
A. brasilense Sp245
PSLC LPPC PSLC LPPC EPS LPS
Specific and nonspecific interactions
R1
Nonspecific interaction A. lipoferum 59b PSLC A. lipoferum RG20a
LPPC LPPC PSLC
A. leguminosarum 252
LPPC PSLC
LII
– A. brasilense Sp7 A. brasilense SR75 – A. brasilense Sp245 A. lipoferum 59b
PSLC LPPC
A. lipoferum RG20a A. leguminosarum 252
Nonspecific interaction PSLC LPPC
A. lipoferum RG20a
Specific and nonspecific interactions
– A. brasilense Sp245 – A. brasilense Sp7
PSLC LPPC
EPS LPS
A. lipoferum 59b
EPS – R. leguminosarum 252
Specific and nonspecific interactions PSLC PSLC LPPC LPPC
A. brasilense Sp7 A. brasilense SR75 A. brasilense Sp245 – A. brasilense S17
A. brasilense S17
Specific interaction PSLC LPPC
LPS – A. brasilense S17 PSLC – R. leguminosarum 252
Nonspecific interaction PSLC LPPC
A. brasilense Sp7 A. brasilense SR75 A. brasilense Sp245
LPPC
R2
PSLC LPPC LPS
A. brasilense S17 – R. leguminosarum 252
Nonspecific interaction PSLC LPPC
A. brasilense Sp7
PSLC LPPC
A. brasilense SR75
PSLC LPPC
A. brasilense Sp245
PSLC LPPC
A. lipoferum 59b
PSLC LPPC
A. lipoferum RG20a
EPS
– R. leguminosarum 252
A. brasilense S17
Fig. 4. Schematic representation of the interactions of the B. polymyxa 1460 and R. leguminosarum 252 lectins with the polysaccharide-containing complexes of azospirilla and rhizobia.
Dot immunoblotting analysis showed (Fig. 3) that the minimum concentration of agglutinin R1 necessary for its interaction with the LPPC of A. brasilense Sp7 is 0.3 µg/ml (the LPPC concentration in this experiment was 67.5 µg/ml). Figure 4 schematically shows the specific and nonspecific interactions of the B. polymyxa 1460 and R. leguminosarum 252 lectins with the polysaccharidecontaining complexes of rhizobia and azospirilla. The specific interaction of the galactose-specific lectin with polysaccharides was also described by Behhary et al. [12] with respect to its involvement in bacterial cell aggregation. Thus, the surface lectins of soil bacteria can interact with the polysaccharide complexes of other soil bacteria liberated into the medium. Such interactions may be
responsible for the formation of nitrogen-fixing bacterial associations in the plant rhizosphere. The blocking of the binding sites of bacterial lectins by the polysaccharide complexes of rhizobia and azospirilla may contribute to their competition for binding sites on the host plant roots. REFERENCES 1. Kijne, J.W., Schaal van der, A.M., Diaz, C.L., and Iron, F., Mannose-Specific Lectins and the Recognition of Pea Roots by Rhizobium leguminosarum, Lectins: Biology, Biochemistry, and Clinical Biochemistry, Berlin, 1983, vol. 3, pp. 521–529. 2. Loh, J.T., Ho, S.C., Wang, J.L., and Schindler, M., Carbohydrate Binding Activities of Bradyrhizobium japonicum. IV. Effect of Lactose and Flavones on the ExpresMICROBIOLOGY
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